半固体培养法制备非洲猪瘟病毒pA104R蛋白的单克隆抗体  

Preparation of monoclonal antibody against pA104R protein of African swine fever virus by semi-solid culture method

作　　者：刘蓓蓓[1,2] 韦艳娜 陈蓉[1,2] 谢星 倪博[1,2] 郝飞 张珍珍 白昀[1,2] 袁厅 冯志新[1,2] LIU Bei-bei;WEI Yan-na;CHEN Rong;XIE Xing;NI Bo;HAO Fei;ZHANG Zhen-zhen;BAI Yun;YUAN Ting;FENG Zhi-xin(Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences/Key Laboratory of Veterinary Biological Enginering and Technology,Ministry of Agriculture and Rural Affairs,Nanjing 210014,China;GuoTai(Taizhou)Center of Technology Innovation for Veterinary Biologicals,Taizhou 225300,China)

机构地区：[1]江苏省农业科学院兽医研究所/农业农村部兽用生物制品工程重点实验室,江苏南京210014 [2]兽用生物制品<泰州>国泰技术创新中心,江苏泰州225300

出　　处：《江苏农业学报》2024年第4期682-689,共8页Jiangsu Journal of Agricultural Sciences

基　　金：国家重点研发计划政府间国际科技创新合作重点专项(2019YFE0107300);江苏省农业科技自主创新基金项目[CX(22)2037]。

摘　　要：为了快速、高效制备非洲猪瘟病毒(ASFV)单克隆抗体,本研究通过大肠杆菌系统表达并纯化了ASFV重组蛋白pA104R。以ASFV重组蛋白pA104R为抗原,分别比较了CpG ODN联合氢氧化铝佐剂和常规弗氏佐剂两种免疫策略,并重点比较半固体培养法和常规有限稀释法来制备ASFV pA104R单克隆抗体的效率。结果显示,本研究获得了相对分子质量为3.5×104的ASFV重组可溶性蛋白pA104R,通过其与CpG ODN联合氢氧化铝佐剂免疫小鼠,在第21 d即可达到融合要求,本试验方法(重组蛋白pA104R与CpG ODN联合氢氧化铝佐剂免疫)较重组蛋白pA104R与常规弗氏佐剂免疫节省14 d时间。通过半固体培养法筛选单克隆的试验周期比有限稀释法缩短28 d,并减少了亚克隆的工作量。半固体培养法获得5株阳性杂交瘤细胞,挑选效价较高的3株(9A4、9H6、11F5)进行鉴定,重链均为IgG,轻链均为KAPPA。纯化后的3株单克隆抗体针对pA104蛋白和全病毒蛋白质的效价分别达1∶160000~1∶320000和1∶200~1∶400。本研究优选了CpG ODN联合氢氧化铝佐剂结合半固体培养法筛选pA104R的单克隆抗体,为单克隆抗体制备提供了快速高效的新策略。In order to prepare the monoclonal antibodies against African swine fever virus(ASFV)rapidly and efficiently,the recombinant pA104R protein of ASFV was systematically expressed and purified by Escherichia coli in this study.The recombinant pA104R protein of ASFV was used as antigen to compare the two immune strategies of CpG ODN combined with aluminum hydroxide adjuvant and conventional Freund’s adjuvant,respectively,and the efficiency of semi-solid culture method and conventional limited dilution method in the preparation of monoclonal antibody against ASFV pA104R was compared.The results showed that the recombinant pA104R soluble protein of ASFV with a relative molecular weight of 3.5×104 was obtained,and the fusion requirement was achieved on the 21st day after the mice were immunized with CpG ODN and aluminum hydroxide adjuvant.Compared with routine Freund’s adjuvant immunization and recombinant pA104R protein,CpG ODN combined with aluminum hydroxide adjuvant and semi-solid culture method in this experiment saved 14 days.The semi-solid culture method shortened the test period of monoclonal screening by 28 days compared with the limited dilution method,and saved a lot of subclonal workload.Five positive hybridoma cell lines were obtained by semi-solid culture method,and three high titer hybridoma cell lines(9A4,9H6,11F5)were selected for identification.The heavy chain was IgG,and the light chain was KAPPA.The titers of three purified monoclonal antibodies against pA104 protein and whole virus protein were 1∶160000-1∶320000 and 1∶200-1∶400,respectively.In this study,monoclonal antibodies were screened by CpG combined with aluminum hydroxide adjuvant and semi-solid culture method,providing a new rapid and efficient strategy for monoclonal antibody preparation.

关 键 词：非洲猪瘟病毒 pA104蛋白 单克隆抗体 半固体培养法 

分 类 号：S852.651[农业科学—基础兽医学]